Research method based on principle of action of low-intensity focused ultrasound on joint stiffness

Abstract

The invention discloses a research method based on the action principle of low-intensity focused ultrasound on joint stiffness, and relates to the technical field of joint stiffness treatment. Safe and effective treatment measures are found for patients with ankylosis; comprising the following steps: experimental animals are grouped and modeled, male rats in specific age groups and weight ranges are selected and randomly divided into a normal group, a stiffness group and an ultrasonic stiffness group, and a joint stiffness model is established; and performing LIFU treatment, namely performing low-intensity focused ultrasound treatment on the rats of the ultrasonic stiffness group. Through qRT-PCR (quantitative reverse transcription-polymerase chain reaction), Western blotting, cellular immunofluorescence and other technical means, the research finds that the LIFU can improve the joint stiffness condition by inhibiting the activation of fibroblasts and the expression of a TGF-beta / Smad signal channel, so that not only is the molecular mechanism of the LIFU for treating the joint stiffness disclosed, but also an important index is provided for evaluating the treatment effect and predicting the disease progress.

Description

Technical Field

[0001] The present invention relates to the technical field of joint stiffness treatment, and in particular to a research method based on the effect principle of low-intensity focused ultrasound on joint stiffness. Background Art

[0002] Joint stiffness is common in patients who need to be immobilized due to musculoskeletal diseases or patients whose activities are limited due to neurological diseases. It seriously affects the patients' lives and reduces their mobility and quality of life. Currently, surgery, drugs and continuous passive movements are mainly used to treat joint stiffness, but the effects are poor or the side effects are large. There is an urgent need to find safe and effective treatment measures.

[0003] Low-intensity focused ultrasound (LIFU) is a focused ultrasound technology that is a safe, non-invasive physical therapy. Previous studies have found that joint stiffness is significantly improved after ultrasound treatment. However, there are few studies on the effect of LIFU in treating joint stiffness, and the mechanism is still unclear. LIFU may improve joint stiffness by controlling the production of inflammatory mediators, inhibiting the expression of fibrosis genes, changing the expression of hypoxia genes, and improving the elasticity of the joint capsule.

[0004] Therefore, this project uses a rat knee stiffness model to analyze the changes in the range of motion and histopathological changes of the joint capsule, combined with molecular biology, cell experiments and genome sequencing, to study the effects and mechanisms of LIFU in the treatment of joint stiffness, in order to enrich and improve the understanding of LIFU and joint stiffness, and provide a theoretical basis for related clinical treatments. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a research method based on the effect of low-intensity focused ultrasound on joint stiffness.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A research method based on the effect of low-intensity focused ultrasound on joint stiffness, comprising the following steps:

[0008] S1: 8-week-old male rats were selected as experimental animals, with body weight controlled at 250-300g. The rats were randomly divided into three groups: normal group, stiff group and ultrasonic stiff group, with 5 rats in each group. The joint stiffness model was established using the flexion internal fixation model. After anesthesia, incisions were made on the outer side of the left thigh and the anterior medial side of the calf of the rat, and the femur and tibia were separated. Holes were punched in the middle section respectively, and sterilized nylon thread was passed through the punched parts and tightened to bind and fix the femur and tibia in a flexed position of about 145°. Finally, the wound was disinfected and the skin was sutured. Three days after the operation, penicillin was injected into the outer thigh muscle of the rat every day to prevent infection. After 4 weeks of fixation, the fixed nylon thread was removed. If the joint range of motion was limited by more than 30°, the model was successfully established.

[0009] S2: LIFU treatment, rats in the ultrasound stiffness group were treated with low-intensity focused ultrasound;

[0010] S3: Cell extraction, culture and grouping: knee joint synovial tissue was isolated from neonatal mice for culture, and normal group, normal + ultrasound group, stiff group and stiff + ultrasound group were set up for cell experiments;

[0011] S4: Joint range of motion measurement: passive joint range of motion of each group of animals was measured before, during and after ultrasound treatment;

[0012] S5: qRT-PCR and Western blotting analysis were performed to detect the expression of fibrosis-related genes and proteins;

[0013] S6: Cell immunofluorescence to observe the expression and localization of fibrosis-related proteins.

[0014] Preferably: in step S1, a flexion internal fixation model is used to establish a joint stiffness model, and the specific steps include anesthesia, incision, separation of femur and tibia, punching, binding and fixation, disinfection and suturing. After fixation for 4 weeks, the fixation nylon thread is removed. If the joint range of motion is limited by more than 30°, the model is successfully established.

[0015] Further: In step S2, low-intensity focused ultrasound therapy was performed using the HIFU Medical Animal Irradiation Ultrasound Test System, with a specific ultrasound frequency and power set, a specific treatment time per day, and a specific number of treatments per week. After the set time was reached, all rats were killed and their left knee joints were collected.

[0016] Further: In step S3, the synovial tissue is digested with type I collagenase, the tissue pieces are filtered and centrifuged, and the cells are cultured in alpha-minimal essential medium supplemented with fetal bovine serum and penicillin-streptomycin.

[0017] As a preferred solution of the present invention: in step S3, in the cell experiment, it is mainly divided into four groups, including a normal group, a normal + ultrasound group, a stiff group and a stiff + ultrasound group. In the cell experiment, the normal + ultrasound group only underwent LIFU treatment, the stiff group only added TGF-β1, and the stiff + ultrasound group underwent LIFU treatment after adding TGF-β1.

[0018] As a further solution of the present invention: in step S4, the axis of the instrument is placed close to the axis of motion of the knee joint, the fixed arm is parallel to the long axis of the femur, and the movable arm is parallel to the long axis of the tibia. A specific torque is used to measure the passive joint motion of each group of animals to avoid damaging the soft tissue around the joint, and the average value is taken as the measurement result.

[0019] As a further embodiment of the present invention: in step S5, qRT-PCR analysis of α-SMA, COL1A, and COL3A1 is performed, and Western blotting analysis is performed using specific antibodies.

[0020] Based on the previous protocol: In step S6, incubation was performed with antibodies against α-SMA, COL1A1, and P-smad2 / 3, and observation was performed by fluorescence microscopy.

[0021] Based on the above scheme: This method also includes analyzing the effect of LIFU on the range of motion of rat joints and the improvement of LIFU on the degree of joint fibrosis.

[0022] Based on the above scheme: This method also includes analyzing the effect of LIFU on synovial fibroblasts and whether LIFU inhibits fibroblast activation by inhibiting the expression of TGF-β / SMAD pathway.

[0023] The beneficial effects of the present invention are:

[0024] 1. A research method based on the effect of low-intensity focused ultrasound on joint stiffness. Through qRT-PCR, Western blotting and cell immunofluorescence, this study found that LIFU can regulate the expression of fibrosis-related factors such as α-SMA, TGF-β1 and COL1A1, which not only helps to reveal the molecular mechanism of LIFU treatment of joint stiffness, but also provides an important indicator for evaluating treatment effects and predicting disease progression.

[0025] 2. A research method based on the effect of low-intensity focused ultrasound on joint stiffness, which reveals the therapeutic effect and mechanism of LIFU on joint stiffness and provides new ideas and methods for the clinical treatment of joint stiffness.

[0026] 3. A research method based on the effect of low-intensity focused ultrasound on joint stiffness. LIFU treatment can reduce the degree of fibrosis in rat joints, which is manifested as lower joint capsule thickening and fibrosis compared with the untreated group. It provides new ideas and methods for the treatment of joint fibrosis and helps to slow down or reverse the process of fibrosis.

[0027] 4. A research method based on the effect of low-intensity focused ultrasound on joint stiffness, using LIFU to inhibit the expression of fibrosis-related genes at the transcriptional and translational levels, thereby inhibiting the activation of myofibroblasts.

[0028] 5. A research method based on the effect of low-intensity focused ultrasound on joint stiffness, using nylon thread for modeling. Compared with the previous modeling methods using Kirschner wires, non-absorbable sutures, etc., Kirschner wires are expensive and non-absorbable, and the success rate of suture modeling is not high. After switching to nylon thread, the cost is low and the success rate is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is an animal modeling flow chart and ROM change diagram of a research method based on the effect principle of low-intensity focused ultrasound on joint stiffness proposed by the present invention (a animal modeling and treatment flow chart; b ROM measurement diagram; c ROM change (A: stiffness group; L: ultrasound group; ns: no statistical significance; *: P≤0.05));

[0030] Figure 2 It is a schematic diagram of animal histological detection of a research method based on the effect principle of low-intensity focused ultrasound on joint stiffness proposed by the present invention (HE and MASSON staining of animal tissue sections and immunohistochemical staining of a-SMA, TGF-B1 and COL1A1);

[0031] Figure 3 It is a schematic diagram of the detection of fibroblast activation based on a research method proposed in the present invention based on the effect of low-intensity focused ultrasound on joint stiffness (ab a-SMA, COL1A1 and COL3A1 gene WB and statistical graphs; ca-SMA and COL1A1 gene RT-qPCR statistical graphs (C: normal group; L: LIFU group; T: TGF-B1 group: THL: TGF-β1+LIFU group; ns: not statistically significant: *: P≤0.05;)).

[0032] Figure 4This is a schematic diagram of the activation of the TGF-B / SMAD pathway based on a research method proposed in the present invention that is based on the effect of low-intensity focused ultrasound on joint stiffness (bc: P-smad2 / 3, smad2 / 3 and smad4 gene WB and statistical graphs (C: normal group; L: LIFU group; T: TGF-B1 group; T+L: TGF-B1+LIFU group; ns: not statistically significant; *: P≤0.05)). DETAILED DESCRIPTION

[0033] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0034] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0035] Embodiment 1:

[0036] A research method based on the effect of low-intensity focused ultrasound on joint stiffness, comprising the following steps:

[0037] S1: Experimental animal grouping and modeling:

[0038] Eight-week-old Sprague Dawley male rats were selected as experimental animals, with a body weight of 250-300 g. The rats were randomly divided into three groups: a normal group, a stiff group, and an ultrasonic stiff group, with 5 rats in each group. A flexion internal fixation model was used to establish a joint stiffness model. The specific steps included: after anesthesia, an incision was made on the outer side of the left thigh and the anterior medial side of the calf of the rat, the femur and tibia were separated, holes were punched in the middle section, sterilized nylon thread was passed through the punched part and tightened, so that the femur and tibia were tied and fixed at a flexion position of about 145°, and finally the wound was disinfected and the skin was sutured. Three days after the operation, penicillin was injected into the outer thigh muscle of the rat every day to prevent infection. After 4 weeks of fixation, the fixed nylon thread was removed. If the joint range of motion was limited by more than 30°, the model was successfully established.

[0039] S2: LIFU treatment:

[0040] A total of 15 Sprague Dawley male rats were used and randomly divided into three groups: normal group, stiff group and ultrasound stiff group, with 5 rats in each group. The normal group did not receive any treatment, while the stiff group and ultrasound group were used to model joint stiffness. After the sutures were removed and the model was successfully established 4 weeks later, the stiff group did not receive any other treatment, while the ultrasound group received low-intensity focused ultrasound (LIFU) treatment after anesthesia. The animal experiment LIFU used the Haifu Medical Animal Irradiation Ultrasound Test System, with an ultrasonic frequency of 0.84Mhz and an ultrasonic power of 1W, 20 minutes a day, 5 times a week, for 4 weeks. Except during anesthesia, all rats moved freely in the cage. After 4 weeks, all rats were killed and the left knee joints were collected.

[0041] S3: Cell extraction, culture and grouping:

[0042] Two-week-old Sprague Dawley suckling mice were selected and killed by cervical dislocation. The synovial tissues of bilateral knee joints were isolated and cut into 1 mm3 pieces. The synovial tissues were digested with type I collagenase (BioFroxx, Germany) at 37°C for 1 hour. The tissue blocks were filtered and centrifuged, and then cultured in alpha-minimum essential medium (MEM-α, Gibco) supplemented with 10% fetal bovine serum (FBS, Pronox) and 1% penicillin-streptomycin (Gibco). All cells were cultured at 37°C and 5% CO2. When the cells grew to about 90% confluence, they were passaged every 2-3 days.

[0043] In the cell experiment, the cells were mainly divided into four groups, including the normal group, the normal + ultrasound group, the stiff group and the stiff + ultrasound group. TGF-β is the strongest profibrotic cytokine. The cells in the normal group were not treated in any way, the normal + ultrasound group was only treated with LIFU, the stiff group was only added with TGF-β1 (10ng / ml), and the stiff + ultrasound group was treated with LIFU 24 hours after the addition of TGF-β1 (10ng / ml). The cell experiment LIFU used the Haifu Medical Cell Irradiation Ultrasonic Test System, with an ultrasonic frequency of 1Mhz, an ultrasonic power of 1W, a 20% duty cycle, and 20 minutes each time.

[0044] S4: Joint range of motion measurement:

[0045] The passive range of motion of the joints of the three groups of animals was measured on the 0th, 14th and 28th day of ultrasound treatment. The axis of the instrument was placed close to the axis of motion of the knee joint, the fixed arm was parallel to the long axis of the femur, and the movable arm was parallel to the long axis of the tibia. All measurements were made with a torque of 4 Ncm to stretch the knee joint to its physiological limit while avoiding damage to the soft tissue around the joint. The measurements were repeated three times and the average value was taken.

[0046] S5: qRT-PCR:

[0047] qRT-PCR was performed for α-SMA, COL1A1, and COL3A1;

[0048] S6: Western blotting:

[0049] Antibodies against α-SMA (Zhengneng Biotechnology, 380653), COL3A1 (Zhengneng Biotechnology, R23957), COL1A1 (Zhengneng Biotechnology, R26615), Phospho-Smad2 / 3 (Zhengneng Biotechnology, 251795), Smad2 / 3 (Zhengneng Biotechnology, 382472), and Smad4 (Zhengneng Biotechnology, R27290) were used for incubation, and GAPDH was used as the internal reference protein. The intensity of the protein bands was analyzed by Image J software;

[0050] S7: Cell immunofluorescence:

[0051] Antibodies against α-SMA, COL1A1, and P-smad2 / 3 were incubated overnight at 4°C and observed by fluorescence microscopy;

[0052] Experimental results:

[0053] After conducting research according to the above research method, the following experimental conclusions were obtained:

[0054] (1) LIFU can improve the joint mobility of rats;

[0055] (2) LIFU can reduce joint fibrosis in rats:

[0056] The results of HE and Masson staining showed that the posterior capsule of the joint was thickened and the fibrosis degree was deepened in both the ultrasound group and the stiff group compared with the normal group. However, the thickening and fibrosis degree of the knee joint capsule in the ultrasound group was lighter than that in the stiff group. In immunohistochemical staining, it was found that the expression of fibrosis-related indicators α-SMA and TGF-β1 in the stiff group was increased compared with the normal group, and after ultrasound treatment, the expression of α-SMA and TGF-β1 was reduced compared with the stiff group.

[0057] (3) LIFU can inhibit the activation of myofibroblasts:

[0058] To explore the effect of LIFU on synovial fibroblasts, primary rat synovial fibroblasts were cultured in vitro, TGF-β1 was used to promote the in vitro profibrotic expression of synovial fibroblasts, and LIFU intervention was performed using the HIFU Medical Cell Irradiation Ultrasound Test System. These results showed that LIFU could inhibit the expression of fibrosis-related genes α-SMA, COL1a1, and COL3a1 at the transcriptional and translational levels;

[0059] (4) LIFU may inhibit fibroblast activation by inhibiting the expression of TGF-β / SMAD pathway:

[0060] Activation of the TGF-β / Smad signaling pathway plays a role in the initiation and development of fibrosis, especially Smad2, Smad3 and Smad4. This study analyzed the effect of LIFU on the expression of TGF-β / Smad signaling pathway proteins in synovial fibroblasts in vitro. These results showed that LIFU could inhibit the expression of the TGF-β / SMAD pathway.

[0061] The above is a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the field within the technical scope disclosed by the present invention in combination with the prior art or public common sense, within the spirit and principle of the present invention, shall be covered by the protection scope of the present invention.

Claims

1. A research method based on the effect of low-intensity focused ultrasound on joint stiffness, characterized in that: The following steps are involved: S1: 8-week-old male rats were selected as experimental animals, with body weight controlled at 250-300g. The rats were randomly divided into three groups: normal group, stiff group and ultrasonic stiff group, with 5 rats in each group. The joint stiffness model was established using the flexion internal fixation model. After anesthesia, incisions were made on the outer side of the left thigh and the anterior medial side of the calf of the rat, and the femur and tibia were separated. Holes were punched in the middle section respectively, and sterilized nylon thread was passed through the punched parts and tightened to bind and fix the femur and tibia in a flexed position of about 145°. Finally, the wound was disinfected and the skin was sutured. Three days after the operation, penicillin was injected into the outer thigh muscle of the rat every day to prevent infection. After 4 weeks of fixation, the fixed nylon thread was removed. If the joint range of motion was limited by more than 30°, the model was successfully established. S2: LIFU treatment, rats in the ultrasound stiffness group were treated with low-intensity focused ultrasound; S3: Cell extraction, culture and grouping: knee joint synovial tissue was isolated from neonatal mice for culture, and normal group, normal + ultrasound group, stiff group and stiff + ultrasound group were set up for cell experiments; S4: Joint range of motion measurement: passive joint range of motion of each group of animals was measured before, during and after ultrasound treatment; S5: qRT-PCR and Western blotting analysis were performed to detect the expression of fibrosis-related genes and proteins; S6: Cell immunofluorescence to observe the expression and localization of fibrosis-related proteins.

2. A research method based on the effect of low-intensity focused ultrasound on joint stiffness according to claim 1, characterized in that: In step S1, a flexion internal fixation model was used to establish a joint stiffness model. The specific steps included anesthesia, incision, separation of femur and tibia, punching, binding and fixation, disinfection and suturing. After fixation for 4 weeks, the fixation nylon thread was removed. If the joint range of motion was limited to more than 30°, the model was successful.

3. A research method based on the effect of low-intensity focused ultrasound on joint stiffness according to claim 2, characterized in that: In step S2, low-intensity focused ultrasound therapy was performed using the HIFU Medical Animal Irradiation Ultrasound Test System. A specific ultrasound frequency and power were set, and treatment was performed for a specific time each day and a specific number of times each week. After the set time was reached, all rats were killed and their left knee joints were collected.

4. A research method based on the effect of low intensity focused ultrasound on joint stiffness according to claim 3, characterized in that: In step S3, synovial tissue is digested with type I collagenase, the tissue pieces are filtered, centrifuged, and the cells are cultured in alpha-minimal essential medium supplemented with fetal bovine serum and penicillin-streptomycin.

5. A research method based on the effect of low intensity focused ultrasound on joint stiffness according to claim 4, characterized in that: In step S3, in the cell experiment, it was mainly divided into four groups, including normal group, normal + ultrasound group, stiff group and stiff + ultrasound group. In the cell experiment, the normal + ultrasound group only underwent LIFU treatment, the stiff group only added TGF-β1, and the stiff + ultrasound group underwent LIFU treatment after adding TGF-β1.

6. A research method based on the effect of low intensity focused ultrasound on joint stiffness according to claim 5, characterized in that: In step S4, the axis of the instrument was placed close to the axis of motion of the knee joint, the fixed arm was parallel to the long axis of the femur, and the movable arm was parallel to the long axis of the tibia. A specific torque was used to measure the passive joint range of motion of each group of animals to avoid damaging the soft tissue around the joint, and the average value was taken as the measurement result.

7. A research method based on the effect of low intensity focused ultrasound on joint stiffness according to claim 6, characterized in that: In step S5, qRT-PCR analysis of α-SMA, COL1A1, and COL3A1 was performed, and Western blotting analysis was performed using specific antibodies.

8. A research method based on the effect of low intensity focused ultrasound on joint stiffness according to claim 7, characterized in that: In step S6, incubation was performed with antibodies against α-SMA, COL1A1, and P-smad2 / 3, and observation was performed using a fluorescence microscope.

9. A research method based on the effect of low intensity focused ultrasound on joint stiffness according to claim 8, characterized in that: The method also includes analyzing the effect of LIFU on the range of motion of rat joints and the improvement of LIFU on the degree of joint fibrosis.

10. A research method based on the effect of low intensity focused ultrasound on joint stiffness according to claim 9, characterized in that: The method also includes analyzing the effect of LIFU on synovial fibroblasts and whether LIFU inhibits fibroblast activation by inhibiting the expression of the TGF-β / SMAD pathway.

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